Skin barrier preparation and method therefor

ABSTRACT

A method of selecting a skin barrier system suitable for infants and young children is disclosed.

This application claims priority of the benefits of the filing of U.S.Provisional Application Ser. No. 62/613,878, filed Jan. 5, 2018, thecontents of which are hereby incorporated by reference herein in theirentirety.

FIELD

The present invention relates to the development of skin barriersystems, particularly for infants or young children, while assessing thelevel of skin protection through analyses of adult skin tests. Theinvention allows one to evaluate the protection level of a skin barriersystem with objective data while avoiding the need to test on youngchildren or infants.

BACKGROUND

Skin cleansers contain surfactants, which may compromise the integrityof skin barrier to the penetration of external aggressors, resulting inskin irritation. Assessing cleanser mildness on skin is typically doneby clinical evaluation and measurement of alterations in trans epidermalwater loss (TEWL) following exaggerated patch test or exaggerated washtest protocols. These methods are partly subjective and often withvariable results.

Skin care product mildness (particularly for cleansing products thatcontain potentially irritating surfactant systems) is typically assessedin vivo in adults using normal-use tests, exaggerated (repeated) usetests or patch tests. Even for baby products, the assessment is firstdone in adults and once passed it is sometimes followed with normal-usetests in infants. Mildness is evaluated as lack of irritation (typicallyskin erythema (redness)). The effects on the skin barrier are typicallyassessed instrumentally by measurements of Trans-Epidermal-Water-Loss(TEWL). The effects of products on the skin barrier may also be studiedex vivo using Franz cells and measuring skin impedance.

However, the previous methods suffer from a number of defects andconcerns. For example, normal use tests typically require large panelsizes in order to differentiate between varying levels of mildness,which can become expensive and time-consuming. In patch tests, thesurfactants can respond differently under occluded versus normal useconditions. The results of arm immersion tests can be weather-dependent.In flex wash tests, the tested skin site may not be representative ofother areas of the body.

Further, each of the above items is evaluated subjectively (clinicalobservation), which may introduce variabilities. Finally, the majorityof these tests are either geared toward adult skin without adequatecorrelation to infant skin, or these studies are performed oninfants/young children, and clinical studies on infants raises ethicaland technical questions. As noted, the validity of directly transferringdata acquired on adults to the case of infant skin has been questioned.For example, infant skin is not typically used in Franz cells and thetransfer of these data to infant skin is questionable. These methods areused always involving a margin of safety factor (typically 10 times)that reflects the uncertainty.

It would be useful to develop a method that can evaluate the impact of asurfactant system on infant and/or young child skin barrier byobjectively assessing the concentration profile of a marker (such ascaffeine) that penetrated into the skin of adult subjects. The presentinvention seeks to evaluate the impact through the use of biomarkertesting on adult skin, using a computational model to evaluate theimpact on infant/young skin, and developing a surfactant system as aresult of these tests and analyses.

Moisturizers are mixtures of chemical agents specially designed to makethe external layers of the skin or hair softer. Personal carecompositions having moisturizing properties are known. Consumers expectsuch compositions to satisfy a range of requirements. Apart from theskin/hair-care effects which determine the intended application, valueis placed on such diverse parameters as dermatological compatibility,appearance, sensory impression, stability in storage and ease of use.Another benefit provided by many moisturizers is protection of the skinfrom exposure to external environment and agents.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the observation of the absorption of exogenouslyapplied water via Raman confocal microspectroscopy 10 seconds afterwater application to the skin of the lower ventral arm.

FIG. 2 shows a comparison between experiment (adult), model (adult) andprediction (baby).

FIG. 3 shows a comparison between experiment, model and prediction forWater and SLS, on adult and infant skin

FIG. 4 shows a comparison between several surfactant formulations.

FIG. 5 shows modeling experimental data in the Adult epidermal model, insilico.

FIG. 6 shows predictive caffeine permeation curves following surfactanttreatment.

FIG. 7 shows predictive absorbed amount in baby Stratum Corneum, Areaunder the Curve for 0-10 μm of depth (mmol caffeine/g keratin).

FIG. 8 shows experimental data on adult skin.

FIG. 9 shows modelization adult skin.

FIG. 10 shows predictive results on infant skin.

FIG. 11 shows experimental data on adult skin.

FIG. 12 shows modelization on adult skin.

FIG. 13 shows predictive results on infant skin.

DETAILED DESCRIPTION

The present invention relates to a process for assessing the mildness ofa skin care product on infant skin and specifically the effect oftopical application of a substance and/or formulation on the skinbarrier of infants, and preparing and/or using a surfactant system basedupon this evaluation. As used herein, the term “infant skin” refers toskin of newborn human children, but also refers to and includes skin ofchildren that are up to 12 months old. The term “young child” or youngchildren” refers to infants but also includes children that are 12 to 36months old.

The purpose of the present invention is to be able to assess productsafety, mildness, etc. on the skin of infants and/or young children bysafely evaluating the product on adult skin. The process involvesapplying the substance on adult skin, collecting penetration data of amarker on the treated adult skin, transferring the information to acomputational model of adult skin, extracting penetration parametersfrom this model, transferring the parameters to a computational model ofinfant skin and visualizing the penetration of the marker in infant skinmodel and drawing conclusions about the effects of the topical producton the infant skin. Ultimately the process then includes preparing asurfactant system as a result of this evaluation, and/or using asurfactant system based upon this evaluation and ultimate preparation ofthe system.

U.S. Published Application No. 20150285787 to Laboratoires Expansciencediscloses a method for identifying at least one biological marker ofchildren's skin that comprises: a) measuring the level of expression ofa candidate biological marker in at least one sample of skin cells (A),said sample being obtained from a donor under 16 years of age, b)measuring the level of expression of said candidate biological marker inat least one control sample (B) of skin cells, c) calculating the ratiobetween the level of expression of step a) and the level of expressionof step b), and d) determining whether the candidate marker is abiological marker of children's skin.

WO2015150426 and WO2017103195 to Laboratoires Expanscience disclosemethods of evaluating in vivo formulations that comprise a) contactingan active agent or a formulation with a reconstructed skin model, saidmodel being obtained from a skin sample from a child; b) contacting thereconstructed skin model after step a) with urine; and c) measuring theexpression level of at least one of a list of specified biologicalmarkers in the skin model after step b.

U.S. Published Application No. 20180185255 to Procter & Gamble disclosesa method of screening cleansers for mildness, comprising: a) measuringthe level of one or more ceramides on an area of skin prior toapplication of a cleanser; b) applying the cleanser to the area of skinfor at least 7 days; c) measuring the level of one or more ceramidesafter the product application of at least 7 days on the area of skin;wherein the cleanser is mild if the level of the one or more ceramidesis at least 10% vs. the no treatment control.

U.S. Pat. No. 10,036,741 to Procter & Gamble discloses a method forevaluating the influence of a perturbagen on skin homeostasis andformulating a skin care composition comprising the perturbagen thatcomprises causing a computer processor to query a data architecture ofstored skin instances associated with a perturbagen with an unhealthyskin gene expression signature, wherein the query comprises comparingthe unhealthy skin gene expression signature to each stored skininstance and assigning a connectivity score to each instance.

EP 1248830A1 to Procter & Gamble discloses the use of a forearmcontrolled application test to assess surfactant mildness.

Saadatmand et al., Skin hydration analysis by experiment and computersimulations and its implications for diapered skin, Skin Res. Technol.,2017: 1-14, discloses a stratum corneum reversible hydration model thatsimulates evaporative water loss and stratum corneum thickness as afunction of exposure scenarios such as time-dependent relative humidity,air temperature, skin temperature and wind velocity.

Maxwell et al., Application of a systems biology approach for skinallergy risk assessment, Proc. 6^(th) World Congress on Alternatives &Animal Use in Life Sciences, pp. 381-388 (2007) discloses an in silicomodel of skin sensitization induction to characterize and quantify thecontribution of each pathway to the overall biological process.

Strube et al., The flex wash test: a method for evaluating the mildnessof personal washing products, J. Soc. Cosmet. Chem., 40:297-306 (1989),discloses the use of a sixty second wash, three times daily, of the flexarm to assess potential irrancy of washing products.

Keswick et al., Comparison of exaggerated and normal use techniques foraccessing the mildness of personal cleansers, J. Soc. Cosmet. Chem.,43:187-193 (1992), discloses the comparison of the forearm test and flexwash test to home use to determine how well the tests approximate ad libusage.

Frosch et al., Journal of the American Academy of Dermatology, Volume 1,Issue 1, 35-41 (1979), discloses a chamber test to assess the irritancyof soaps that entails five weekday exposures to 8% solutions withreadings of scaling and redness.

Many in vivo tests are not acceptable for experimental use on infantskin. The cited references do not disclose or suggest the evaluation ofadult skin and the use of computational models to correlate how aningredient would affect infant skin. The invention thus avoids the needto conduct an in vivo test on infant skin.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety herein. As used herein, allpercentages are by weight unless otherwise specified. In addition, allranges set forth herein are meant to include any combinations of valuesbetween the two endpoints, inclusively.

In the present invention, the method can be used to discriminate betweendifferent cleanser formulations according to their effect on skinbarrier to external penetration. The present invention sets forth amethod of analyzing the formulation that objectively evaluates theeffects of topical cleansers on skin barrier to the penetration ofexternal aggressors and can be used to assess cleanser mildness. One cantake the results of this analysis and provide or prepare a suitableformulation that is considered mild to an infant's and/or a youngperson's skin.

The present invention is drawn to a predictive method for assessing themildness of a topical substance toward the skin of a subject, preferablyyoung infant. It also relates to a predictive method to assess thepenetration of a compound (marker) through the skin of infants. Thepresent invention also relates to a predictive method for assessing theinfluence of a topically applied substance on the penetration of acompound (marker) though infant skin. In addition, the present inventionmay provide a method to measure and/or predict the effect of barrierenhancement of topical substance(s).

In one aspect, the present invention may include a number of processsteps. It may include a Phase 1 (in vivo) and Phase 2 (in silico), withoptional Phase 3 (intellectual process) and finally the processconcludes with the preparation of a suitable surfactant system that hasbeen analyzed to pass the aforementioned tests, or the process concludeswith the application of a surfactant system onto the skin of an infantand/or young child.

Phase I, In Vivo

-   -   A. Applying a topical substance on adult skin, such as through        direct application or application on a patch or other delivery        system.    -   B. Topically apply a marker on the adult skin and collect        penetration data of said marker on the substance treated adult        skin. This step includes applying the marker and then following        its concentration profile through the skin for example using        confocal Raman micro-spectroscopy (CRM).

Phase II, in Silico

-   -   C. Transferring the information (penetration data) to a        computational model of adult skin and extracting penetration        parameters from this model.        -   This step can also be described as using a computational            adult skin penetration model to visualize the penetration of            the marker by optimizing the penetration parameters (for            example local surface concentration and permeability            coefficients) so that the model penetration profiles will            match the experimental data.    -   D. Transferring the penetration parameters (following        appropriate transformations) to a computational model of infant        skin and visualizing the penetration of the marker in infant        skin model.

(Optional), Phase III, Intellectual Process

-   -   E. Drawing conclusions about the mildness (effects) of the        topical product on the infant skin, based on the amount of        marker that has penetrated the baby skin model.

Once the aforementioned process steps have been completed and theconclusions in Step E are made, the surfactant system may be prepared,applied, or distributed by the user.

Topical Substance

The invention includes one or more topical substances to be evaluated,where the topical substance is desired to be used in the finalsurfactant system. The topical substance is any type of substanceapplied on the skin that has an effect on the permeability of thestratum corneum. The topical substance will modify the permeation of themarker through the skin. By measuring the marker permeation, the effectof the topical substance may be assessed. Typically, for the testoutlined above, the topical substance is impregnated on a patch that isheld in contact with the skin for 30 minutes before the marker isapplied. The patch may include one or more topical substances forapplication testing.

Different types of topical substance can be evaluated in the scope ofthe present invention, for example, the topical substance may be a harshsubstance(s) that can decrease the barrier properties of the skin andincrease the permeation of the marker. In this case the presentinvention can allow one to create a mildness classification of thesubstance(s) and help to opt for a milder solution when designing a newskin product composition, without in vivo or in vitro testing. In otheraspects, the topical substance may include a barrier substance(s), thatare designed to help protect the skin and increase its barrier property,thus reducing the permeation of marker though the skin. As noted above,the present invention can help to select the most efficient solutionwithout having to perform in vitro or in vivo testing on infant skin.

Marker

The present invention uses one or more markers or biomarkers in theevaluation method. Any type of marker is suitable, as long as there is amethod to trace the marker and generate a concentration profile (e.g.,penetration data). In the example of using confocal Ramanmicro-spectrometry, the marker should have a traceable signal in theRaman spectrum. In another example a fluorescent marker can be tracedusing confocal fluorescence microscopy. The penetration kinetics of themarker ideally should be such that a steady state of concentrationprofile is reached within reasonable time (for example in up to onehour).

The marker can be hydrophilic, lipophilic or amphoteric which willdefine what type of barrier effect the evaluator is examining. Forexample, one suitable marker is caffeine. In the case of caffeine, theanalysis examines the barrier to hydrophilic substances.

A marker according to the present invention may include any moleculethat is safe toxicologically and dermatologically, has reasonablepenetration kinetics and is traceable by confocal analysis.

-   -   Safety; some markers used in the past are not acceptable due to        toxicity reasons (Dansyl Chloride (proposed in Paye et al.        “Dansyl chloride labelling of stratum corneum: its rapid        extraction from skin can predict skin irritation due to        surfactants and cleansing products” Contact Dermatitis 30(2),        91-96, 1994) has been discontinued due to risk of sensitization        and skin corrosion upon skin contact).    -   Penetration kinetics; a molecule that penetrates the skin, e.g.,        is fast enough but not too fast. E.g., a molecule that has a        permeability coefficient close to caffeine may be employed:        kp=1.16×10⁻⁴ cm/h reported in Dias M et al. “Topical delivery of        caffeine from some commercial formulations” Int J Pharm        1999182(1): 41-7    -   Confocal analyses are noninvasive and provide data about depth        penetration of markers. In contrast, for example, tape stripping        is invasive and destroys the barrier; this is not acceptable in        the present invention.

Penetration Data

The present invention analyzes penetration data. Penetration data is aconcentration profile; this means the concentration of the marker as afunction of depth through the skin. The present invention may use anydesired analytical method suitable to measure the concentration profileof a marker as a function of depth in the skin and more precisely in theepidermis and particularly the Stratum Corneum. Any desired methods maybe used, while non-invasive methods are preferred. Confocal techniquesare preferred because they are noninvasive and provide reasonableresolution, for example 3 to 5 μm resolution in the perpendicular toskin surface direction, up to 200 μm in depth. One such method includesconfocal Raman micro-spectrometry, but other methods, including confocalfluorescence microscopy, may be used.

Computational Model of Adult/Infant Skins

The present invention uses computational models to evaluate thecomponents of the surfactant system tested. Any model that can result ina concentration profile of a marker through skin may be employed. Theuser may choose any type of computational skin penetration model that,given the penetration parameters, can result in a concentration profileof a marker through the skin. The use of both adult skin and skin modelsrequires that the models take into account the structure of the skinarchitecture and the differences there exist between the two.

For example, one can use the physiological model published in Sutterlinet al., “A 3D self-organizing multicellular epidermis model of barrierformation and hydration with realistic cell morphology based on EPISIM”,Scientific Reports, volume 7, article 43472, 2017; with modification tointegrate substances (e.g., the marker) diffusion though the skinlayers. Sutterlin et al. discloses a cell behavioral model (CBM)encompassing regulatory feedback loops between the epidermal barrier,water loss to the environment, and water and calcium flow within thetissue. The EPISIM platform consists of two ready-to-use software tools:(i) EPISIM Modellar (graphical modelling system) and (ii) EPISIMSimulator (agent-based simulation environment). Each EPISIM-based modelis composed of at least a cell behavioral and a biomechanical model (CBMand BM). The BM covers all spatial and biophysical cell properties. CBMsare models of cellular decisions. A 2D or 3D version of the model may beused in accordance with the invention (a version of the 2D model, butwithout the stratum corneum component, is described in: Suetterlin etal. “Modeling multi-cellular behavior in epidermal tissue homeostasisvia finite state machines in multi-agent systems”, Bioinformatics,25(16), 2057-2063, 2009.

In one method, the process begins by the user letting the simulationreach a steady state corresponding to epidermal homeostasis. Then, at agiven timepoint corresponding to the topical application of the marker,the evaluator introduces a user-defined variable corresponding to theskin surface concentration (C_(surface)) of the marker. The value ofthis parameter is defined from the concentration profile obtainedexperimentally and corresponds to the marker concentration at depth 0(skin surface). A cell variable is introduced to the model defining theconcentration of the marker in the cell (C_(cell)). At each time thisparameter is modified based on the Fick's law of diffusion, as themarker is allowed to diffuse from each cell to its immediate neighbors.To apply Fick's law, a permeability coefficient parameter (P) isintroduced in the model. This permeability coefficient parameterinherently accounts for the diffusion coefficient, the resistance todiffusion due to a partition coefficient and the resistance to diffusiondue to the distance of the path that the substance has to cross to gofrom one cell to the next. The permeability coefficient is different forthe stratum corneum (P_(SC)) compared to the viable epidermis (P_(VE)).If the substance reaches the lowest part of the epidermis, it is allowedto diffuse to the dermal compartment which is modeled as a penetration“sink”.

These modifications apply both to the adult skin and the infant skinmodel.

The infant skin model is created by modifying the parameters of theadult model to reflect the higher turnover rate (proliferation anddesquamation) in baby skin.

Penetration Parameters

Penetration parameters are characterizing the penetration kinetics, howeasy it is for a substance to cross the surface and go deep in the skin.It can be, for example, the partition coefficient, the diffusioncoefficient, and/or the permeability coefficient.

Mildness Index

At steady state, the concentration profile of the marker in the adultskin model is compared to the experimental concentration profile. If theprofiles do not match, the penetration parameters (C_(surface), P_(SC)and P_(VE)) are adjusted and the simulation is repeated. Once the twoprofiles match, then the parameters are used to calculate thecorresponding parameters for the marker penetration in the infant skinmodel. Due to higher hydrophilicity of the baby skin the C_(surface)parameter is higher (typically twice that of the adult skin*), whereasthe other penetration parameters remain the same between the two models.*See, e.g., Nikolovski et al., Barrier function and water-holding andtransport properties of infant stratum corneum are different from adultand continue to develop through the first year of life, Journal ofInvestigative Dermatology (2008), Vol. 128, which uses tools such astransepidermal water loss (TEWL), skin capacitance,absorption-desorption, and Raman confocal spectroscopy to demonstratethat the water storing and water transport properties of the stratumcorneum of infants is different than that for adults. In particular, thereference discloses observation of the absorption of exogenously appliedwater via Raman confocal microspectroscopy 10 seconds after waterapplication to the skin of the lower ventral arm.

FIG. 5 a therein (and reproduced in FIG. 1A) shows that a significantamount of water absorption was found in the stratum corneum of infantsless than 12 months old. FIG. 5 b therein (and reproduced in FIG. 1B)shows that, in contrast, no significant water absorption was found inadult skin after water application. It is expected that caffeinepermeation, which is highly hydrophilic, would behave similarly to waterpermeation.

Then, the marker penetration is allowed to reach steady state at theinfant skin model (about 1000 steps with each step corresponding to 30min physiology time). At steady state, the average concentration profileof the marker is calculated (average concentration as a function ofdepth). The area under the curve (AUC, integral) is calculated for theconcentration profile down to a defined depth (such as 20 μm).

A mildness index scale can be defined from the AUC values correspondingto different product treatments. This is an arbitrary scale used toclassify the mildness of the topical substances.

This mildness index value allows the evaluator to compare the mildnessof the tested topical substance with respect to two referencesubstances; water (mild) and sodium lauryl sulfate (SLS) 0.1% (harsh).With water and SLS 0.1% as the two reference points it is possible tobuild a scale to measure mildness of other topical substances.

It should be noted that this mildness indicator is optional, and one canomit the mildness indicator and directly compare the relative mildnessof different topical substances directly to one another based on theintegration of their predictive penetration curves (i.e., the calculatedAUC values).

EXAMPLES

1—Comparison Between Experiment (Adult), Model (Adult) and Prediction(Baby). See FIG. 2 .

Goals:

-   -   Show the predictive effect on infant skin of two extreme topical        solutions, one harsh (containing 0.1% SLS), and one mild        (water).    -   Show that the adult model fits with the experimental data.    -   Show that topical substances don't have the same effect on adult        and infant skin, infant skin being more permeable to marker.

Comparison between experiment, model and prediction for Water and SLS,on adult and infant skin. See FIG. 3 3 above shows the depth (in μm) ofpenetration of caffeine (marker) expressed in mmol per gram of keratin,obtained by in vivo experiment on adult skin (lines + and ∘) or insilico predictive model (lines Δ, x, ⋄ and □).

The effect of 2 topical solutions on caffeine penetration is displayedin FIG. 3 , water and 0.1% SLS. Model calculated data for adult arerepresented by lines Δ and ⋄; for water and SLS respectively. Predictivedata for infant are represented by lines x and □, for water and SLSrespectively.

In vivo experimental data is collected on adult skin then transferred inan adult skin model to simulate the caffeine depth of penetration in anadult skin. Prediction of the penetration of caffeine in the infant skinproposed by the present invention model is represented in diagonal crossline (x) when the skin is treated with a water patch prior to caffeineapplication and by a square line (□) when the skin is treated with a0.1% SLS patch prior to caffeine application.

Area under the curve from 0 to 20 μm of skin depth gives an indicationof the level of mildness of the topical substance. The lower the milderto the skin. The area under the curve is a key parameter to be able tocompare different treatment.

2—Comparison Between Several Surfactant Formulations. See FIG. 4 .

Goal:

-   -   Create a predictive surfactant formulation classification based        on their mildness to infant skin.

Step 2.1: Experimental data, caffeine penetration in adult skin, in vivo

Formulations tested are shown in FIG. 4 .

Experimental protocol is as disclosed in the material and method ofarticle Stamatas et al., Development of a non-invasive optical methodfor assessment of skin barrier to external penetration, BiomedicalOptics and 3D Imaging OSA (2012). Stamatas et al. discloses the use ofcharacteristic Raman spectrum of caffeine to track caffeine penetrationthrough adult skin to demonstrate the impact of (1) sodium laurylsulfate and (2) barrier cream on stratum corneum barrier function.

Step 2.2: Modeling experimental data in the Adult epidermal model, insilico. See FIG. 5 .

Experimental caffeine penetration data collected at step 2.1 aretransferred to a computational model of adult skin. Simulation of skinpenetration is performed for each topical substance; a single simulationper substance may be sufficient. Caffeine penetration parameters (localsurface concentration and permeability coefficients) are extracted.

Step 2.3: Predictive caffeine permeation curves following surfactanttreatment. See FIG. 6 . Caffeine penetration parameters obtained fromthe adult skin model at steps 2.2 are transferred to a computationalmodel of infant skin. Simulation of infant skin penetration is performedfor each topical substance. Predictive caffeine penetration results areextracted and displayed in Graph 4 above.

Step 2.4: Predictive absorbed amount in baby Stratum Corneum, Area underthe Curve for 0-10 μm of depth (mmol caffeine/g keratin). See FIG. 7 .Predictive curves for each topical agent displayed in FIG. 6 in step 2.3are integrated to obtain, for each topical agent, a predictive amount ofthe caffeine absorbed in the infant stratum corneum. These values aredisplayed in FIG. 7 .

In other words, this predictive graph shows how much caffeine willpenetrate within the first 10 μm (not mm) of SC. The more caffeine wehave, the more aggressive is the topical substance.

Results

Surprisingly, it can be predicted from this graph that the topical agentwill not have a mildness index value regarding infant skin alwaysreflected by the experimental values obtained on adult skin:

-   -   Formulation 3 will be milder than Formulation 5.    -   Formulation 4 will be milder than Formulation 2.

As a result of this experiment, a composition including the formulationin Formulations 3 and/or 4 can be prepared and applied to the skin ofinfant and/or young skin as preferable to Formulations 5 and 2correspondingly.

In the next embodiment, the invention relates to the development ofbarrier systems, particularly for infants or young children, whileassessing the level of barrier effect through analyses of adult skintests.

The invention allows one to evaluate the protection level of a barriersystem with objective data while avoiding the need to test on youngchildren or infants.

Method

Steps I and II disclosed above remain the same. Predictive data oninfant skin penetration of a marker are generated.

Step III differs in that the data relates to use of low penetration anddiffusion of the marker through the skin to predict the barrier effectthat the topical substance applied in step I A would have on an infantor baby skin.

Leave-on products (e.g., creams/moisturizers) may be assessed using thismethodology

Experiments Example 3

Material & Method

Data on adult skin were collected on healthy volunteers, with normalskin, who agreed not to use any other skincare treatment on the forearmsfor at least 24 hrs before the study and during the study.

Instrument Used:

In vivo Confocal Raman Microspectrometer (Skin Composition AnalyzerModel 3510, River Diagnostics, Rotterdam, The Netherlands)

Caffeine patch: 180 mg of Caffeine in 10 mL of demineralized water, 1.8%

Example 4 Barrier Cream Simulation

Experimental data were collected on 5 female volunteers, aged between 20and 35 years.

Topical Substance Tested:

-   -   Barrier cream: Desitin® Creamy (diaper rash cream)    -   US INCI list: Zinc oxide 10%, inactive ingredients (aloe        barbadensis leaf juice), cyclomethicone, dimethicone, fragrance,        methylparaben, microcrystalline wax, mineral oil, propylparaben,        purified water, sodium borate, sorbitan sesuileate, vitamin E,        white petrolatum, white wax.

Protocol

-   -   1—5 minutes acclimatization in a temperature- and        humidity-controlled room    -   2—Application of the topical substance on the forearm    -   3—30 minutes acclimatization in a temperature- and        humidity-controlled room    -   4—Application of the caffeine patch on forearm (same location)        for 30 minutes    -   5—Measurement in the Raman fingerprint region

Results

1—Experimental Data on Adult Skin

Data from Desitin-treated skins (Square) are compared to data fromreference (Circle) untreated skins (i.e., no topical substance appliedin step 2 of the protocol).

Penetration data are extracted from the experimental results andtransferred in computational model of adult skin.

2—Modelization Adult Skin. See FIG. 9 .

The next step is to define the skin permeation parameters on thecomputational model of the adult skin so that it can accurately simulatethe experimental data presented above.

These parameters are calculated from the slope of the caffeinepenetration profile.

The results from the adult model is shown below.

Barrier cream treated skin (Square) is compared to a reference untreatedskin (Circle). Penetration parameters are extracted from thecomputational adult model.

3—Predictive Results on Infant Skin. See FIG. 10 .

The last step comprises of transferring the caffeine penetrationparameters with appropriate transformations to the infant skincomputational model to simulate the predicted caffeine penetration inthe infant skin.

Predictive results for caffeine penetration on a barrier cream treatedskin (Square) and a reference untreated skin (Circle) are shown below.

Finally, from the ratio shown below involving the area under the curve(AUC) for the Untreated skin by the AUC for the Barrier cream-treatedskin we can calculate the predicted % protection of the barrier cream:% Protection=100×(AUC (Untreated)−AUC (Product))/AUC (Untreated)=89.18%

Examples 5 Moisturizer Simulation

Experimental data were collected on 6 volunteers, aged between 18 and 40years.

Topical Substance Tested:

-   -   Moisturizer A: Emulsion comprising: Glycerin (12%), Petrolatum        (4%), Distearyldimonium Chloride, Water    -   Moisturizer B: Structured emulsion comprising: Petrolatum (40%),        Glycerin (12%), Distearyldimonium Chloride, Water

Protocol

-   -   1—5 minutes acclimatization in temperature/humidity-controlled        room    -   2—Application of the topical substance on forearm for 30 minutes    -   3—Application of the caffeine patch on forearm (same location)        for 30 minutes    -   4—Measurement in the fingerprint region

Results

1—Experimental Data on Adult Skin

Data from Moisturizer A treated skins (Square) are compared to data fromMoisturizer B treated skins (Triangle) and reference untreated skins(Circle) (i.e. no topical substance applied in step 2 of the protocol).

Penetration data are extracted from the experimental results andtransferred in computational model of adult skin.

2—Modelization Adult Skin. See FIG. 12 .

The next step is to define the skin permeation parameters on thecomputational model of the adult skin so that it can accurately simulatethe experimental data presented above.

These parameters are calculated from the slope of the caffeinepenetration profile.

The results from the adult model is shown below.

Moisturizer A treated skin (Square) is compared to Moisturizer B treatedskin (Triangle) and to a reference untreated skin (Circle).

Penetration parameters are extracted from the computational adult model.

3—Predictive Results on Infant Skin

The last step comprises of transferring the caffeine penetrationparameters with appropriate transformations to the infant skincomputational model to simulate the predicted caffeine penetration inthe infant skin.

Predictive results for caffeine penetration on a Moisturizer A treatedskin (Square), a Moisturizer B treated skin (Triangle) and a referenceuntreated skin (Circle) are shown in FIG. 13 .

Finally, from the ratio shown below involving the area under the curve(AUC) for the Untreated skin by the AUC for the moisturizer-treated skinwe can calculate the predicted % protection of the moisturizer:

For Moisturizer A% Protection: Not applicable

-   -   The area under the curve for moisturizer A is superior to the        area under the curve for the untreated reference. The simulation        predicts no protecting effect on infant skin.

For Moisturizer B% Protection=100×(AUC (Untreated)−AUC (Product))/AUC (Untreated)=17.72%.

It will be understood that, while various aspects of the presentdisclosure have been illustrated and described by way of example, theinvention claimed herein is not limited thereto, but may be otherwisevariously embodied according to the scope of the claims presented inthis and/or any derivative patent application.

The invention claimed is:
 1. A non-invasive in vivo method of evaluatinga potential impact of a barrier system on infant skin, comprising: a)topically applying said barrier system in a non-invasive in vivo methodto adult skin; b) topically applying a marker to said barrier systemtreated adult skin; c) applying confocal analysis to measure-penetrationof said marker into said barrier system treated adult skin; d) using acomputational model of adult skin penetration to visualize penetrationof the marker by optimizing penetration parameters so that the model ofadult skin penetration profiles match experimental data; e) transferringthe optimized penetration parameters to a computational model of infantskin; and f) determining the penetration of the marker in thecomputational model of infant skin; wherein step c) comprises followinga concentration profile of said marker through the skin, wherein saidconcentration profile is measured using confocal Ramanmicro-spectroscopy (CRM) or confocal fluorescence microscopy; whereinsaid penetration parameters are selected from the group consisting ofskin surface concentration (Csurface), permeability coefficient for thestratum corneum (PSC) and permeability coefficient for the viableepidermis (PVE); and wherein said penetration parameters are transferredin step e) such that the skin surface concentration (Csurface) parameteris higher in the computational model of infant skin than in thecomputational model of adult skin penetration, and such that thepermeability coefficient for the stratum corneum (PSC) and thepermeability coefficient for the viable epidermis (PVE) are the samebetween the two models.
 2. The method of claim 1, wherein the marker iscaffeine.
 3. The method of claim 1, wherein EPISIM is employed as thecomputational model of adult skin penetration.
 4. The method of claim 1,wherein the computational model of adult skin penetration is anagent-based model.
 5. The method of claim 1, wherein said skin surfaceconcentration (Csurface) parameter in the computational model of infantskin is twice that of the skin surface concentration (Csurface)parameter in the computational model of adult skin penetration.
 6. Themethod of claim 1, wherein said barrier system is topically appliedthrough direct application or through application on a patch.